A variety of methods for the analytic determination of hydrogen peroxide have been known. One class of such methods involves the use of the enzyme peroxidase to catalyze the reaction of a substrate with hydrogen peroxide to oxidize the substrate and form water. Enzymatic determinations of this type have found particular utility in the analysis of various substances, such as cholesterol, glucose and uric acid, in body fluids such as blood. In such methods, the body fluid is admixed with an enzyme capable of catalyzing oxidation of the substance to be determined with the concurrent formation of hydrogen peroxide. The fluid also is admixed with peroxidase and a substrate for peroxidase which, on oxidation, undergoes a color change. The extent of the color change is a measure of the amount of hydrogen peroxide formed, which in turn is a measure of the substance to be determined.
For example, Hall et al, in "Automated Determination of Glucose Using Glucose Oxidase and Potassium Ferrocyanide", Analyt. Biochem., 26, 12-17 (1968), describe a method for the quantitative determination of glucose by oxidation with glucose oxidase to form gluconic acid and hydrogen peroxide, and reduction of the hydrogen peroxide with potassium ferrocyanide in the presence of peroxidase to form ferricyanide. Trinder, in "Determination of Glucose in Blood Using Glucose Oxidase With an Alternative Oxygen Acceptor", Ann. Clin. Biochem., 6, 24-27 (1969), described the use of phenol and 4-aminophenazone (4-aminoantipyrine) as the color-forming system in place of the potassium ferrocyanide. In this system, it is believed that the phenol is oxidized and then reacts with the 4-aminophenazone to form a quinonimine dye; a reaction which previously had been described by Emerson in "The Condensation of Aminoantipyrine II. A New Color Test for Phenolic Compounds", J. Org. Chem., 8, 417-28 (1943) and in U.S. Pat. No. 2,194,201 granted Mar. 19, 1940. Still more recently, Meiattini, in U.S. Pat. No. 3,866,045, granted May 27, 1975 (Re. U.S. Pat. No. 29,498 granted Dec. 20, 1977) applied the reactants described by Emerson to the Trinder reaction.
This type of procedure can be employed to determine other constituents of blood or other body fluids by replacing glucose oxidase with an enzyme capable of catalyzing the oxidation of another component, e.g., cholesterol oxidase or uric acid oxidase (uricase), with concurrent formation of hydrogen peroxide. For example, the adaptation of this procedure to the determination of serum uric acid is described by Trivedi et al in "New Enzymatic Method for Serum Uric Acid at 500 nm", Clin. Chem., 24, (11), 1908-11 (1978). However, the Trinder-type chromophore is not as sensitive as is desired for the determination of metabolites such as uric acid, which ordinarily are present in blood in very low concentrations, e.g., of the order of 4 to 9 mg/100 ml of blood, as compared with concentrations of 80-100 mg/100 ml for glucose and 150-250 mg/100 ml for cholesterol.
Another type of chromogen-chromophore system which has been employed as a hydrogen peroxide detector in analytical procedures of this type is the 3-methyl-2-benzothiazolinone hydrazone/N,N-dimethylaniline system, in which the two reagents oxidatively couple to form a covalently bonded indamine dye. The application of this procedure to the analysis of uric acid and glucose was reported by Gochman et al in "Automated Determination of Uric Acid, With Use of a Uricase-Peroxidase System", Clin. Chem., 17, (12), 1154-59 (1971) and "Application of a New Peroxide Indicator Reaction to the Specific, Automated Determination of Glucose with Glucose Oxidase", Clin. Chem., 18, (9), 943-50 (1972).